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Alfred Wittinghofer

Publications and source records attributed to Alfred Wittinghofer.

45 records · Page 3Linked to original sources
PubMed2003

Biochemical characterization of the Ran-RanBP1-RanGAP system: are RanBP proteins and the acidic tail of RanGAP required for the Ran-RanGAP GTPase reaction?

RanBP type proteins have been reported to increase the catalytic efficiency of the RanGAP-mediated GTPase reaction on Ran. Since the structure of the Ran-RanBP1-RanGAP complex showed RanBP1 to be located away from the active site, we reinvestigated the reaction using fluorescence spectroscopy under pre-steady-state conditions. We can show that RanBP1 indeed does not influence the rate-limiting step of the reaction, which is the cleavage of GTP and/or the release of product P(i). It does, however, influence the dynamics of the Ran-RanGAP interaction, its most dramatic effect being the 20-fold stimulation of the already very fast association reaction such that it is under diffusion control (4.5 x 10(8) M(-1) s(-1)). Having established a valuable kinetic system for the interaction analysis, we also found, in contrast to previous findings, that the highly conserved acidic C-terminal end of RanGAP is not required for the switch-off reaction. Rather, genetic experiments in Saccharomyces cerevisiae demonstrate a profound effect of the acidic tail on microtubule organization during mitosis. We propose that the acidic tail of RanGAP is required for a process during mitosis.

Amino Acid Sequence↗
PubMed2002

Fluorescently labelled guanine nucleotide binding proteins to analyse elementary steps of GAP-catalysed reactions.

Downregulation of small guanine nucleotide-binding proteins (GNBPs) requires the interaction with their corresponding GTPase-activating proteins (GAPs), which increase the slow intrinsic GTPase reaction by several orders of magnitude. On the basis of the structure of H-Ras in complex with the catalytic domain of p120-GAP, we have developed a set of site-specifically labelled Ras-variants, one of which turned out to be particularly sensitive for studying the interaction with Ras-specific GAPs. This specific fluorescent reporter group and the use of manganese to increase the rate of the chemical reaction step allowed us to identify differences in the rate-limiting step of either the GAP-334 or NF1-333 catalyzed reaction. The assay was also applied to study the interaction of the Ras-related protein Rap1B with Rap1GAP, for which no detailed kinetic analysis was available. Single-turnover experiments of this reaction show that the low affinity of the complex (50 microM) is due to a slow association rate as well as a fast dissociation rate. RapGAP promotes AlFx binding to Rap1B, even though it does not contain a catalytic arginine. The rate-limiting step of the RapGAP catalysed reaction is release of inorganic phosphate, which is about five times slower than the chemical cleavage step. Our data reveal marked differences in GAP/target interactions even between closely related systems and suggest that the fluorescent reporter group method might be generally applicable to many other GNBPs and their cognate GAPs.

Amino Acid Substitution↗
PubMed2002

Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE.

The bacterial enteropathogen Salmonella typhimurium employs a type III secretion system to inject bacterial toxins into the host cell cytosol. These toxins transiently activate Rho family GTP-binding protein-dependent signaling cascades to induce cytoskeletal rearrangements. One of these translocated Salmonella toxins, SopE, can activate Cdc42 in a Dbl-like fashion despite its lack of sequence similarity to Dbl-like proteins, the Rho-specific eukaryotic guanine nucleotide exchange factors. To elucidate the mechanism of SopE-mediated guanine nucleotide exchange, we have analyzed the structure of the complex between a catalytic fragment of SopE and Cdc42. SopE binds to and locks the switch I and switch II regions of Cdc42 in a conformation that promotes guanine nucleotide release. This conformation is strikingly similar to that of Rac1 in complex with the eukaryotic Dbl-like exchange factor Tiam1. However, the catalytic domain of SopE has an entirely different architecture from that of Tiam1 and interacts with the switch regions via different amino acids. Therefore, SopE represents the first example of a non-Dbl-like protein capable of inducing guanine nucleotide exchange in Rho family proteins.

Amino Acid Sequence↗
PubMed2002

The complex of Arl2-GTP and PDE delta: from structure to function.

Arf-like (Arl) proteins are close relatives of the Arf regulators of vesicular transport, but their function is unknown. Here, we present the crystal structure of full-length Arl2-GTP in complex with its effector PDE delta solved in two crystal forms (Protein Data Bank codes 1KSG, 1KSH and 1KSJ). Arl2 shows a dramatic conformational change from the GDP-bound form, which suggests that it is reversibly membrane associated. PDE delta is structurally closely related to RhoGDI and contains a deep empty hydrophobic pocket. Further experiments show that H-Ras, Rheb, Rho6 and G alpha(i1) interact with PDE delta and that, at least for H-Ras, the intact C-terminus is required. We suggest PDE delta to be a specific soluble transport factor for certain prenylated proteins and Arl2-GTP a regulator of PDE delta-mediated transport.

Amino Acid Sequence↗
PubMed2002

RanGAP mediates GTP hydrolysis without an arginine finger.

GTPase-activating proteins (GAPs) increase the rate of GTP hydrolysis on guanine nucleotide-binding proteins by many orders of magnitude. Studies with Ras and Rho have elucidated the mechanism of GAP action by showing that their catalytic machinery is both stabilized by GAP binding and complemented by the insertion of a so-called 'arginine finger' into the phosphate-binding pocket. This has been proposed as a universal mechanism for GAP-mediated GTP hydrolysis. Ran is a nuclear Ras-related protein that regulates both transport between the nucleus and cytoplasm during interphase, and formation of the mitotic spindle and/or nuclear envelope in dividing cells. Ran-GTP is hydrolysed by the combined action of Ran-binding proteins (RanBPs) and RanGAP. Here we present the three-dimensional structure of a Ran-RanBP1-RanGAP ternary complex in the ground state and in a transition-state mimic. The structure and biochemical experiments show that RanGAP does not act through an arginine finger, that the basic machinery for fast GTP hydrolysis is provided exclusively by Ran and that correct positioning of the catalytic glutamine is essential for catalysis.

Arginine↗
PubMed2002

Rap-specific GTPase activating protein follows an alternative mechanism.

Rap1 is a small GTPase that is involved in signal transduction cascades. It is highly homologous to Ras but it is down-regulated by its own set of GTPase activating proteins (GAPs). To investigate the mechanism of the GTP-hydrolysis reaction catalyzed by Rap1GAP, a catalytically active fragment was expressed in Escherichia coli and characterized by kinetic and mutagenesis studies. The GTPase reaction of Rap1 is stimulated 10(5)-fold by Rap1GAP and has a k(cat) of 6 s(-1) at 25 degrees C. The catalytic effect of GAPs from Ras, Rho, and Rabs depends on a crucial arginine which is inserted into the active site. However, all seven highly conserved arginines of Rap1GAP can be mutated without dramatically reducing V(max) of the GTP-hydrolysis reaction. We found instead two lysines whose mutations reduce catalysis 25- and 100-fold, most likely by an affinity effect. Rap1GAP does also not supply the crucial glutamine that is missing in Rap proteins at position 61. The Rap1(G12V) mutant which in Ras reduces catalysis 10(6)-fold is shown to be efficiently down-regulated by Rap1GAP. As an alternative, Rap1(F64A) is shown by kinetic and cell biological studies to be a Rap1GAP-resistant mutant. This study supports the notion of a completely different mechanism of the Rap1GAP-catalyzed GTP-hydrolysis reaction on Rap1.

Amino Acid Sequence↗
PubMed2002

Obg, a G domain with a beautiful extension.

The structure of Obg, a protein involved in a complicated genetic network that regulates stress response and sporulation in Bacillus subtilis, reveals a completely new type of guanine nucleotide binding protein and provides some hints about its function.

Bacillus subtilis↗